臺灣博碩士論文加值系統

本研究的目的在探討deleted in azoospermia-like (dazl)和germ cell-less (gcl)基因在黑鯛(Acanthopagrus schlegeli)生殖腺的基因表現。實驗從黑鯛選殖出dazl和gcl全長基因，dazl cDNA全長為2359個核甘酸，可轉譯為200個胺基酸，gcl cDNA全長為3415個核甘酸，可轉譯為503個胺基酸。RT-PCR的結果顯示，dazl只會專一地在生殖腺表現，gcl在生殖腺的表現較強，在腦、眼睛、心臟、肝臟、腎臟等器官的表現較弱，而在肌肉則不會表現。dazl和gcl在120天的表現有顯著的增加，推測dazl和gcl的表現應該和性別分化有關。在孵化後的120、150、180天，E2則會抑制dazl和gcl的基因表現。1+黑鯛dazl在8至12月精巢表現量高於卵巢，推測和精子生成作用有關。精巢切除的2+黑鯛， gcl在1月的表現量有顯著的增加，推測此時gcl表現可能與卵細胞成熟過程有關。免疫組織化學染色的結果，Dazl和Gcl皆表現在卵原細胞、初級卵母細胞、卵黃堆積卵細胞、精原細胞。Dazl還會表現在卵黃堆積卵細胞周圍的濾泡細胞。RNA原位雜交染色顯示，在第一次產卵季節的生殖腺，dazl和gcl會表現在初級卵母細胞。

The purpose of this study was to investigate the gene expresstion of deleted in azoospermia-like (dazl) and germ cell-less (gcl) in the gonad of black porgy, Acanthopagrus schlegeli. dazl full length cDNA contains 2359 nucleotides encoding a putative 200 amino acid (aa) protein, and gcl contains 3415 nucleotides encoding a putative 503 aa protein. By RT-PCR, dazl is specificly expressed only in the gonad, and gcl has a high expression in the gonad and low expression level in brain, eyes, heart, liver, kidney and is not detectable in muscle. The gene expression of dazl and gcl are significantly increased at 120 dah. This suggests that dazl and gcl are closely related to sex differention in black porgy. E2 suppresses the expression of dazl and gcl at 120, 150, 180 dah. The expression of dazl in testicular tissues are higher than ovarian tissues from Aug to Dec in 1+-yr black porgy. This suggests that dazl may be related to spermatogenesis.The expression of gcl in January is significantly increased in the testis-excised gonads of 2+-yr black porgy, suggesting that gcl may be associated with oocyte maturation. By immunohistochemistry, Dazl and Gcl are expressed in oogonia, primary oocyte, vitellogenic oocytes, spermatogonia. Dazl is also expressed in the follicle cells surrounding the vitellogenic oocytes. By RNA in situ hybridization, dazl and gcl were co-expressed in the primary oocytes of the gonad in the first spawning season.

謝辭 i
中文摘要 ii
英文摘要 iii
目錄 iv
圖表目錄 vi
壹、文獻整理 1
一、魚類的性別決定 1
二、魚類的性別分化和發育 1
三、精子生成 (spermatogenesis) 2
四、卵子生成 (oogenesis) 3
五、gcl (germ cell-less) 3
六、dazl (deleted in azoospermia-like) 4
七、黑鯛生殖腺的發育 6
八、實驗緣起與目的 7
貳、實驗材料與方法 8
一、實驗魚種 8
二、實驗設計 8
（1）黑鯛gcl、dazl基因選殖 8
（2）抗體誘導 8
（3）黑鯛生殖腺發育情形 9
（4）0+黑鯛在雌二醇 (estradiol-17β, E2) 處理下，gcl、dazl在生殖腺的基因表現情形 9
（5）1+黑鯛性別發育的過程，gcl、dazl在生殖腺的基因表現 9
（6）2+精巢切除的黑鯛，gcl、dazl在卵巢發育過程的基因表現 10
三、實驗方法 10
四、實驗藥品 21
参、實驗結果 26
一、黑鯛dazl和gcl基因選殖 26
二、黑鯛生殖腺發育情形 27
三、基因表現 27
（1）0+黑鯛在E2處理下，dazl和gcl在生殖腺的基因表現情形 27
（2）1+黑鯛性別發育的過程，dazl和gcl在生殖腺的基因表現 28
（3）dazl和gcl在2+精巢切除的黑鯛，卵巢發育過程的基因表現 28
四、組織染色 29
（1）免疫組織化學染色 (Immunohistochemistry) 29
（2）免疫螢光染色 (Immunofluorescence) 30
肆、討論 31
伍、結論 34
陸、參考文獻 71

李孟芳，2008。黑鯛生殖管系統的發育及調控，國立臺灣海洋大學博士論文。
洪維志，2011。類胰島素生長因子在點帶石斑生殖腺性別分化過程的基因表現，國立臺灣海洋大學碩士論文。
郭俊佑，2010。類胰島素生長因子在黑鯛生殖腺性別分化及性轉變過程的基因表現，國立臺灣海洋大學碩士論文。
黃靜端，2002。黑鯛生殖腺組織及微細構造之特性研究，國立臺灣海洋大學博士論文。
Almeida, F. F. L., Kristoffersen, C., Taranger, G. L., Schulz, R.W., 2008. Spermatogenesis in Atlantic cod (Gadus morhua): a novel model of cystic germ cell development. Biol. Reprod. 78, 27-34.
Aoki, Y., Nagao, I., Saito, D., Ebe, Y., Kinjo, M., Tanaka, M., 2008. Temporal and spatial localization of three germline-specific proteins in medaka. Dev. Dyn. 237, 800-7.
Bachvarova, R. F., Crother, B. I., Manova, K., Chatfield, J., Shoemaker, C. M., Crews, D. P., Johnson, A. D., 2009. Expression of Dazl and Vasa in turtle embryos and ovaries: evidence for inductive specification of germ cells. Evol. Dev. 11, 525-34.
Baker, J., Hardy, M. P., Zhou, J., Bondy, C., Lupu, F., Bellve, A. R., Efstratiadis, A., 1996. Effects of an Igf1 gene null mutation on mouse reproduction. Mol. Endocrinol. 10, 903-18.
Barlow, D. P., Stoger, R., Herrmann, B. G., Saito, K., Schweifer, N., 1991. The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 349, 84-7.
Barouller, J. F., Guiguen, Y., Fostier, A., 1999. Endocrine and environmental aspects of sex differentiation in fish. Cell. Mol. Life Sci. 55, 910-31.
Blázquez, M., González, A., Mylonas, C. C., Piferrer, F., 2011. Cloning and sequence analysis of a vasa homolog in the European sea bass (Dicentrarchus labrax): Tissue distribution and mRNA expression levels during early development and sex differentiation. Gen. Comp. Endocrinol. 170, 322-33.
Braat, A. K., Speksnijder, J. E., Zivkovic, D., 1999a. Germ line development in fishes. Int. J. Dev. Biol. 43, 745-60.
Braat, A. K., van de Water, S., Korving, J., Zivkovic, D., 2001. A zebrafish vasa morphant abolishes Vasa protein but does not affect the
establishment of the germline. Genesis. 30, 183-5.
Braun, R. E., 1998 Post-transcriptional control of gene expression during
spermatogenesis. Semin. Cell. Dev. Biol. 9, 483-9.
Brekhman, V., Itskovitz-Eldor, J., Yodko, E., Deutsch, M., Seligman, J.,
2000. The DAZL1 gene is expressed in human male and female
embryonic gonads before meiosis. Mol. Hum. Reprod. 6, 465-8.
Cardwell, J. R., Liley N. R., 1991. Hormonal control of sex and color change in the stop light parrotfish, Sparisoma viride. Gen. Comp. Endocrinol. 81, 7-20.
Cauffman, G., Van de Velde, H., Liebaers, I., Van Steirteghem, A., 2005. DAZL expression in human oocytes, preimplantation embryos and embryonic stem cells. Mol. Hum. Reprod. 11, 405-11.
Chang, S., Ngoh, G. H., Kueh, L. F. S., Qin, Q. W., Chen, C. L., Lam, T. J., Sin, Y. M., 2001. Development of a tropical marine fish cell line from Asian seabass (Lates calcarifer) for virus isolation. Aquaculture 192, 133-45.
Chen, J., Melton, C., Suh, N., Oh, J. S., Horner, K., Xie, F., Sette, C.,
Blelloch, R., Conti, M., 2011. Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition. Genes Dev. 25, 755-66.
Cooke, H. J., Lee, M., Kerr, S., Ruggiu, M., 1996. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. 5, 513-6.
Crews, D., 1994. Temperature, steroids and sex determination. J. Endocrinol. 142, 1-8.
Deshpande, G., Calhoun, G., Yanowitz, J. L., Schedl, P. D., 1999. Novel functions of nanos in downregulating mitosis and transcription during the development of the Drosophila germline. Cell. 99, 271-81.
Devlin, R. H., Nagahama, Y., 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191-364.
Dupont, J., Dunn, S. E., Barrett, J. C., LeRoith, D., 2003. Microarray analysis and identification of novel molecules involved in insulin-like growth factor-1 receptor signaling and gene expression. Recent Prog. Horm. Res. 58, 325-42.
Eberhart, C. G., Maines, J. Z., Wasserman, S. A., 1996. Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia. Nature. 381, 783-5.
Gill, M. E., Hu, Y. C., Lin, Y., Page, D.C., 2011. Licensing of gametogenesis dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells. Proc. Natl. Acad. Sci. USA. 108, 7443-8.
Huang, J. D., Lee, M. F., Chang, C. F., 2002. The morphology of gonadal tissue and male germ cell in the protandrous black porgy, Acanthopagrus schlegeli. Zool. Stud. 41, 216-27.
Jongens, T. A., Ackerman L. D., Swedlow J. R., Jan L. Y., Jan Y. N., 1994. Germ cellless encodes a cell type-specific nuclear pore-associated protein and functions early in the germ-cell specification pathway of Drosophila.Genes Dev.15, 2123-36.
Jongens T. A., Hay B., Jan L. Y., Jan Y. N., 1992. The germ cell-less gene product: a posteriorly localized component necessary for germ cell developmentin Drosophila. Cell. 21, 569-84.
Joshi, R. L., Lamothe, B., Cordonnier, N., Mesbah, K., Monthioux, E., Jami, J., Bucchini, D., 1996. Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J. 15, 1542-7.
Kimura, T., Ito, C., Watanabe, S., Takahashi, T., Ikawa, M., Yomogida, K., Fujita, Y., Ikeuchi, M., Asada, N., Matsumiya, K., Okuyama, A., Okabe, M., Toshimori, K., Nakano, T., 2003. Mouse germ cell-less as an essential component for nuclear integrity. Mol. Cell. Biol. 23, 1304-15.
Kimura, T., Yomogida, K., Iwai, N., Kato, Y., Nakano, T., 1999. Molecular cloning and genomic organization of mouse homologue of Drosophila germ cell-less and its expression in germ lineage cells. Biochem. Biophys. Res. Commun. 262, 223-30.
Kleiman, S. E., Yogev, L., Gal-Yam, E. N., Hauser, R., Gamzu, R., Botchan, A., Paz, G., Yavetz, H., Maymon, B. B. S., Schreiber, L., Barzilai, S., Amariglio, N., Rechavi, G., Simon, A. J., 2003. Reduced human germ cell-less (HGCL) expression in azoospermic men with severe germinal cell impairment. J. Androl. 24, 670-5.
Kobayashi, T., 2010. In vitro germ cell differentiation during sex differentiation in a teleost fish. The International J. Dev. Biol. 54, 105-11.
Kobayashi, T., Kajiura-Kobayashi, H., Nagahama, Y., 2002. Two isoforms of vasa homologs in a teleost fish: their differential expression during germ cell ifferentiation. Mech. Dev. 111, 167-71.
Kosaka, K., Kawakami, K., Sakamoto, H., Inoue, K., 2007. Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis. Mech. Dev. 124, 279-89.
Krøvel, A. V., Olsen, L. C., 2004. Sexual dimorphic expression pattern of a splice variant of zebrafish vasa during gonadal development. Dev. Biol. 271, 190-7.
Le Gac, F., Loir, M., le Bail, P. Y., Ollitrault, M., 1996. Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells. Mol. Reprod. Dev. 44, 23-35.
Leatherman, J. L., Kaestner, K. H., Jongens, T. A., 2000. Identification of a mouse germ cell-less homologue with conserved activity in Drosophila. Mech. Dev. 92, 145-53.
Leatherman, J. L., Levin, L., Boero, J., Jongens, T. A., 2002. Germ cell-less acts to repress transcription during the establishment of the Drosophila germ cell lineage. Curr. Biol. 12, 1681-5.
Le Roith, D., Zick, Y., 2001. Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care. 24, 588-97.
Lee, M. F., Huang, J. D., Chang, C. F., 2008. Development of ovarian tissue and female germ cells in the protandrous black porgy, Acanthopagrus schlegeli (Perciformes, Sparidae). Zool. Stud. 47,
302-16.
Li, M., Shen, Q., Xu, H., Wong, F. M., Cui, J., Li, Z., Hong, N., Wang, L., Zhao, H., Ma, B., Hong, Y., 2011. Differential conservation and divergence of fertility genes boule and dazl in the rainbow trout. PLoS One. 6, 1-12.
Li, W. Y., Deng, F. J., Wang, H. N., Zhen, Y., Xiang, F., Sui, Y., Li, J. Z., 2006. Germ cell-less expression in zebrafish embryos. Dev. Growth Differ. 48, 333-8.
Louvi, A., Accili, D., Efstratiadis, A., 1997. Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev. Biol. 189, 33-48.
Lubzens, E., Young, G., Bobe, J., Cerdà, J., 2010. Oogenesis in teleosts: how eggs are formed. Gen Comp Endocrinol.165(3), 367-89.
Maures, T., Chan, S. J., Xu, B., Sun, H., Ding, J., Duan, C., 2002. Structural, biochemical, and expression analysis of two distinct insulin-like growth factor I receptors and their ligands in zebrafish. Endocrinology. 143, 1858-71.
Michael, J. W., Xu, E., 2011. The roles of the DAZ family in spermatogenesis More than just translation? Spermatogenesis. 1, 36-46.
Miyake, A., Saito, T., Kashiwagi, T., Ando, D., Yamamoto, A., Suzuki, T., Nakatsuji, N., Nakatsuji, T., 2006. Cloning and pattern of expression of the shiro-uo vasa gene during embryogenesis and its roles in PGC development. Int. J. Dev. Biol. 50, 619-25.
Moore, J., Han, H., Lasko, P., 2009. Bruno negatively regulates germ cell-less expression in a BRE-independent manner. Mech. Dev. 126, 503-16.
Nagai, Y., 1992. Primary sex determination in mammals. Zool. Sci. 9, 475-98.
Nagahama, Y., 2005. Molecular mechanisms of sex determination and gonadal sex differentiation in fish. Fish Physiol. Biochem. 31, 105-9.
Nakamura, M., Bhandari, R. K., Higa, M., 2003. The role estrogens play in sex differentiation and sex changes of fish. Fish Physiol. Biochem. 28, 113-7.
Nakamura, K., Fujiwara, H., Higuchi, T., Honda, T., Yamada, S., Nakayama, T., Fujita, J., Maeda, M., Tachibana, T., Suginami, H., Mori, T., 1998. Bestatin, an aminopeptidase inhibitor, promotes follicular growth and ovulation suppressed by stress in mice. J. Endocrinol. 45, 547-53.
Nanda, I., Kondo, M., Hornung, U., Asakawa, S., Winkler, C., Shimizu, A., Shan, Z. H., Haaf, T., Shimizu, N., Shima, A ., Schmid, M., Schartl, M., 2002. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc. Nati. Acad. Sci. USA. 99, 11778-83.
Navarro, I., Leibush, B., Moon, T. W., Plisetskaya, E. M., Banos, N., Mendez, E., Planas, J. V., Gutierrez, J., 1999. Insulin, insulin-like growth factor-I (IGF-I) and glucagon: the evolution of their receptors. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 122, 137-53.
Newton, H. B., 2003. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. Part 1: Growth factor and Ras signaling pathways. Expert Rev Anticancer Ther. 3, 595-614.
Pelegri, F., Knaut, H., Maischein, H. M., Schulte-Merker, S., Nusslein -Volhard, C., 1999. A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization. Curr. Biol. 9, 1431-40.
Perrot, V., Moiseeva, E. B., Gozes, Y., Chan, S. J., Funkenstein, B., 2000. Insulin-like growth factor receptors and their ligands in gonads of a hermaphroditic species, the gilthead seabream (Sparus aurata): expression and cellular localization. Biol. Reprod. 63, 229-41.
Raghuveer, K., Senthilkumaran, B., 2010. Cloning and differential expression pattern of vasa in the developing and recrudescing gonads of catfish, Clarias gariepinus. Comparative Biochem. Physiol. Part A: Mol. Integrative Physiol. 157, 79-85.
Reijo, R., Lee, T. Y., Salo, P., Alagappan, R., Brown, L. G., Rosenberg, M., 1995. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet. 10, 383-93.
Rich, J. N., Bigner, D. D., 2004. Development of novel targeted therapies in the treatment of malignant glioma. Nat. Rev. Drug Discov. 3, 430-46.
Ruggiu M., Speed R., Taggart M., McKay S. J., Kilanowski F., Saunders P.
T., 1997. The mouse Dazla gene encodes a cytoplasmic protein
essential for gametogenesis. Nature. 389, 73-7.
Sakai, F., Kobayash, T., Matsuda, M., Nagahama, Y., 2008. Stability in aromatase immunoreactivity of steroid-producing cells during early development of XX gonads of the Nile tilapia, Oreochromis niloticus: an organ culture study. Zool. Sci. 25, 344-8.
Scholz, S., Domaschke, H., Kanamori, A., Ostermann, K., Rodel, G., Gutzeit, H. O., 2004. Germ cell-less expression in medaka (Oryzias latipes). Mol. Reprod. Dev. 67, 15-8.
Schulz, R. W., de França, L. R., Lareyre, J. J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R. H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol. 165(3), 390-411.
Smith, C. A., Sinclair, A. H., 2004. Sex determination: insights from the chicken. BioEssays 26, 120-32.
Taniguchi, C. M., Kondo, T., Sajan, M., Luo, J., Bronson, R., Asano, T., Farese, R., Cantley, L. C., Kahn, C. R., 2006. Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKClambda/zeta. Cell Metab. 3, 343-53.
Tobler, H., 1986. The differentiation of germ and somatic cell lines in nematodes. Results Probl. Cell Differ. 13, 1-69.
Ullrich, A., Gray, A., Tam, A. W., Yang-Feng, T., Tsubokawa, M., Collins, C., Henzel, W., Le Bon, T., Kathuria, S., Chen, E., et al., 1986. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 5, 2503-12.
Vangompel, M. J., Xu, E.Y., 2011. The roles of the DAZ family in spermatogenesis: More than just translation? Spermatogenesis 1, 36-46.
Wallace R. A., Selman, K., 1981. Cellular and dynamic aspects of oocyte growth in teleosts. Amer. Zool. 21, 325-43.
Wallace R. A., Selman, K., 1990. Ultrastructural aspects of oogenesis and oocyte growth in fish and amphibians. J. Electr. Microsc. Techn. 16, 175-201.
Wu, G. C., Du, J. L., Lee, Y. H., Lee, M. F., Chang, C. F., 2005. Current status of genetic and endocrine factors in the sex change of protandrous black porgy, Acanthopagrus schlegeli (Teleostean). Ann. NY. Acad. Sci. 1040, 206-14.
Xu, H., Gui, J., Hong, Y., 2005. Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev. Dyn. 233, 872-82.
Xu, E. Y., Moore, F. L., Pera, R. A., 2001. A gene family required for
human germ cell development evolved from an ancient meiotic gene conserved in metazoans. Proc. Natl. Acad. Sci. 98, 7414-19.
Yao, H. H., 2005. The pathway to femaleness: current knowledge on embryonic development of the ovary. Mol. Cell. Endocrinol. 230, 87-93.
Yoon, C., Kawakami, K., Hopkins, N., 1997. Zebrafish vasa homologue RNA is localized to the cleavage planes of 2-and 4-cell-stage embryos and is expressed in the primordial germ cells. Development. 124, 3157-63.
Yu, Z., Ji, P., Cao, J., Zhu, S., Li, Y., Zheng, L., Chen, X., Feng, L., 2009. Dazl promotes germ cell differentiation from embryonic stem cells. J Mol. Cell Biol. 1, 93-103.